Bryoid compositions, methods of making and use thereof

ABSTRACT

Embodiments of the present invention feature novel Bryoid compositions, methods of making and methods of treating disease.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/647,237, filed May 26, 2015, which is a 371 U.S. National Phase ofInternational Application No.: PCT/US2013/72070, filed Nov. 26, 2013,which claims the benefit of U.S. Provisional Patent Application No.61/730,227, filed Nov. 27, 2012, the entire contents of which areincorporated herein by reference.

STATEMENT REGARDING FEDERAL SPONSORSHIP

The inventions of the present application were developed with Federalsponsorship under National Institute of Aging and National Institutes ofHealth Grant Number SR44AG034760.

FIELD OF INVENTION

Embodiments of the present invention are directed to compositions havingutility as therapeutics in neurodegenerative diseases such as HutchinsonDisease, Parkinson's disease, Down's syndrome and Alzheimer's disease,virus latency diseases such as HIV and Herpes, cancers such as prostateand other amyloid mediated diseases such as glaucoma.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases, such as Alzheimer's disease, Hutchinson'sDisease, Parkinson's disease, Kuru, Creutzfeldt-Jakob disease and otherspongiform encephalopathies remain major health problems. Currentlythere are very limited means to treat these diseases. With respect toAlzheimer's, Hutchinson's and Parkinson's diseases, these diseases tendto manifest themselves in older individuals and as the diseasesprogress; the afflicted individuals are less able to care forthemselves. The neurogenerative diseases are associated with theformation of beta amyloid plaques. Bryostatin 1 stimulates theproduction of certain isoforms of protein kinase C (PKC) that increasethe production of alpha-secretase which makes soluble amyloid precursorprotein, thus inhibiting the formation of beta amyloid plaques, Withrespect to cancers such as prostate cancer, Bryostatin 1 inhibitsphorbol ester-induced apoptosis in prostate cancer cells bydifferentially modulating protein kinase C (PKC) delta translocation andpreventing PKCdelta-mediated release of tumor necrosis factor-alpha.With respect to virus latency diseases such as HIV latency,Bryostatin-1, as well as many PKC agonists, activates cellulartranscription factors such as NF-kB that binds the HIV-1 promoter andregulates its transcriptional activity. In HIV-1 latency the viralpromoter is less accessible to cellular transcription factors becausenuclear histones surrounding the viral promoter are deacetylated(compacted chromatin). Thus HDAC inhibitors may increase the aceytationof histones (relaxed chromatin) and then transcription factors may havean easy access to the HIV promoter.

Bryoids consist of a family of bryostatins that are complex cyclicmacrolide molecules. Bryoids were originally isolated from the marinebryozoan, Bulgula neritina, in small quantities. Methods of synthesisare awkward and costly. About twenty Bryoid compositions, known asbryostatins and numbered 1-20, have been identified. Many of the bryoidsare known to possess anti-cancer properties.

It would be useful to have new Bryoid compounds that possess highpotency and activity.

SUMMARY OF THE INVENTION

Embodiments of the present invention feature a first Bryoid compositionhaving a molecular weight of approximately 896-898 Amu (Mass+Sodium)having a purity of approximately 50% to a crystal forming purity. Thefirst Bryoid composition can also be characterized as a Bryoid compoundhaving a molecular weight of approximately 873-875 Amu (monoisotopicmass) having a purity of approximately 50% and a crystal forming purity.The first Bryoid composition has a measured mass plus sodium of 897.2Amu and a measured monoisotopic mass of 874.2 Amu. The detaileddiscussion which follows will refer to this Bryoid as B10.

Embodiments of the present invention feature a second Bryoid compositionhaving a molecular weight of approximately 910-912 Amu (Mass+Sodium)having a purity of approximately 50% to a crystal forming purity. Thesecond Bryoid composition can also be characterized as a Bryoid compoundhaving a molecular weight of approximately 888-890 Amu (monoisotopicmass) having a purity of approximately 50% and a crystal forming purity.The second Bryoid composition has a measured mass plus sodium of 911.5Amu and a measured monoisotopic mass of 888.9 Amu. The detaileddiscussion which follows will refer to this Bryoid as B12.

Embodiments of the present invention feature a third Bryoid compositionhaving a molecular weight of approximately 868-870 Amu (Mass+Sodium)having a purity of approximately 50% to a crystal forming purity. Thethird Bryoid composition can also be characterized as a Bryoid compoundhaving a molecular weight of approximately 846-848 Amu (monoisotopicmass) having a purity of approximately 50% and a crystal forming purity.The third Bryoid composition has a measured mass plus sodium of 869.5Amu and a measured monoisotopic mass of 846.6 Amu. The detaileddiscussion which follows will refer to this Bryoid as B14B.

Embodiments of the present invention feature a fourth Bryoid compositionhaving a molecular weight of approximately 895-897 Amu (Mass+Sodium)having a purity of approximately 50% to a crystal forming purity. Thefourth Bryoid composition can also be characterized as a Bryoid compoundhaving a molecular weight of approximately 872-874 Amu (monoisotopicmass) having a purity of approximately 50% and a crystal forming purity.The fourth Bryoid composition has a measured mass plus sodium of 895.5Amu and a measured monoisotopic mass of 872.6 Amu. The detaileddiscussion which follows will refer to this Bryoid as B14C.

These Bryoid compounds of the present invention have molecular weightsthat are different than the molecular weights of bryostatins 1-20.

As used herein, crystal forming purity means the composition has apurity which enables the composition to form crystals. Normally, suchpurity is greater than 90%, and more often greater than 95% purity.Examples presented in this application feature compositions having apurity greater than 99%. Crystal purity would comprise compositions inwhich no impurities can be detected, but is not so limited.

The Bryoid composition of the present invention has utility in thetreatment of Bryoid responsive conditions such as neurodegenerativediseases, cancers and virus latencies. The Bryoid composition of thepresent invention is highly active modulators of certain isoforms ofprotein kinase C (PKC) and amyloid precursor protein. The Bryoids, andthe Bryoid composition of the present invention, stimulate theproduction of certain isoforms of protein kinase C (PKC) that increasethe production of alpha (alpha) secretase which transforms amyloidprecursor protein into soluble forms. Bryoids composition of the presentinvention exhibit high levels of activity similar to or greater thanbryostatin 1.

One embodiment of the present invention is directed to the treatment ofa disease such as a neurodegenerative disease, cancer and virus latencyresponsive to Bryoids, such as Bryostatins 1-20. The method comprisesthe step of administering an effective amount of at least one bryoidcomposition selected from the group consisting of the first Bryoidcomposition, the second Bryoid composition, the third Bryoidcomposition, and the fourth Bryoid composition.

Embodiments of the present invention further comprise the Bryoidcomposition selected from the group consisting of the first Bryoidcomposition, the second Bryoid composition, the third Bryoidcomposition, and the fourth Bryoid composition in a dosage form foradministration to a patient. The dosage form may take many formsincluding without limitation, intravenous, intraperitoneal, oral dosageforms, such as tablets, gel caps, capsules, oral solutions andsuspensions; aerosols, such as spray or mist forming solutions foradministration to lungs, or nasal passageways, topical forms such asointments, lotions, patches and sprays; and other dosage forms known inthe art.

A further embodiment of the present invention is directed to a method ofmaking a Bryoid composition selected from the group consisting of thefirst Bryoid composition, the second Bryoid composition, the thirdBryoid, and the fourth Bryoid composition comprising the steps ofisolating the a Bryoid composition from a source of Bryoids andpurifying the Bryoid composition to a purity of 50% and a crystalforming purity. The source of Bryoids is preferably the marine bryozoan,Bugula neritina.

These and other features and advantages of the present invention will beapparent upon viewing the Figures and reading the detailed descriptionsthat follow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a high performance liquid chromatography scan ofBryostatin 1;

FIG. 2 depicts a HPLC Chromatogram of B. neritina Ethyl Acetate (EA)crude extract;

FIG. 3 depicts a flow chart of purifying steps for Bryostatin-typecompositions;

FIG. 4 depicts alpha-secretase activity induced by Bryostatin-1 andother extracts which embody aspects of the present invention;

FIG. 5 depicts a chromatogram of a mixture of Bryoids;

FIG. 6 depicts a chromatogram of a mixture of Bryoids and identifiesretention times monitored at 265 nm;

FIG. 7 depicts UV spectra of different Bryoids at 265 nm;

FIG. 8-1 depicts a mass spectrum of Bryostatin 1, scanning from 700-1000Amu;

FIG. 8-2 depicts a mass spectrum of a fraction with an internalidentifications 104 and B08 associated with Bryostatin 2, scanning from700-1000 Amu;

FIG. 8-3 depicts a mass spectrum of a fraction with internaldesignations 106 and B14 associated with Bryostatin 3, scanning from700-1000 Amu;

FIG. 8-4 depicts a mass spectrum of a fraction with internaldesignations 112 and B16 embodying features of the present invention,scanning from 700-1000 Amu;

FIG. 8-5 depicts a mass spectrum of a fraction with internaldesignations 102 and B12 and B14 associated with Bryostatin-3, scanningfrom 700-1000 Amu;

FIG. 8-6 depicts a mass spectrum of a fraction with internaldesignations 103 and B10 and B12, scanning from 700-1000 Amu;

FIG. 8-7 depicts a mass spectrum of a fraction with internaldesignations 105 and B12, and B14 associated with Bryostatin-3 scanningfrom 700-1000 Amu;

FIG. 9 depicts UV spectra of the first Bryoid composition and the secondBryoid composition;

FIG. 10 depicts the effect of Bryostatin-1 and different Bryoids at10-⁹M on alpha-secretase activity in SHSY-5Y neuroblastoma cells;

FIG. 11 depicts the effect of Bryostatin-1 and different Bryoids at10-⁹M on PKC-epsilon activity in SHSY-5Y neuroblastoma cells;

FIG. 12 depicts the effect of Bryostatin-1 and different Bryoids at10-⁹M on PKC-delta activity in SHSY-5Y neuroblastoma cells;

FIG. 13 depicts the effect of Bryostatin-1 and different Bryoids at10-⁹M on PKC-alpha activity in SHSY-5Y neuroblastoma cells;

FIG. 14 depicts the proposed structure of a first Bryoid;

FIG. 15 depicts the proposed structure of a second Bryoid;

FIG. 16 depicts the NMR spectra of Bryostatin-3;

FIG. 17 depicts the NMR spectra of the first Bryoid; and,

FIG. 18 depicts the NMR spectra of the second Bryoid.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will now be described with respectto a Bryoid composition selected from the group consisting of the firstBryoid composition (sometimes referred to as B10), the second Bryoidcomposition (sometimes referred to as B12), the third Bryoid composition(sometimes referred to as B14B), the fourth Bryoid composition(sometimes referred to as B14C). These Bryoid compounds of the presentinvention have molecular weights that are different than the molecularweights of Bryostatins 1-20, with the exception of B12 which appears tobe a stereoisomer of Bryostatin 3.

Bugula neritina was fractionated to produce Bryostatin fractions(Bryoids) and isolate individual Bryoids.

HPLC Analysis:

Bryostatin-1 was analyzed by HPLC using a 15 cm 5 micron Phenomenex LunaPFP (2) column (UPS Packing L43) and a mobile phase of 60% acetonitrileacidified with 50 microliters of 85% H₃PO₄ per liter. The flow rate wasset to 1.0 mL per minute and the column temperature was set at 30° C. AWaters Millennium system incorporating a Model 996 photodiode arraydetector was used to generate the chromatographic scans (FIG. 1).Bryostatin-1 was monitored at 265 nm, and contour plots weresimultaneously reported from 195 nm to 345 nm.

Bryostatin-1 Manufacturing and Characterization:

In the first two steps, Bryostatins are extracted from wet Bugulaneritina with organic solvents including isopropanol, methanol, ethylacetate and water followed by silica chromatography using mobile phasesconsisting of hexane/methylene chloride and ethyl acetate/methanol oralternatively extracted from washed, dried and milled Bugula neritinawith SuperFluids™ (near-critical and supercritical fluids with orwithout cosolvents) carbon dioxide and methanol and partially purifiedby SuperFluids™ silica chromatography with carbon dioxide and methanol(Castor, 1998, 2001).

The third step is a segmentation chromatography step on a CG71 polymericresin (Rohm-Haas) with a mobile phase consisting of methanol and waterthat improves the purity of Bryostatin-1 to 60-70%. The fourth steputilizes a segmentation chromatographic method using two semi-prep HPLCC18 columns (Baker Scientific, Phenomenex) with a mobile phaseconsisting of acetonitrile and water to improve the Bryostatin-1 purityto >95%. The fifth step utilizes crystallization with acetonitrile andwater to purify Bryostatin-1 to >98.5%.

The identity of the Bryostatin-1 product was confirmed by Ultra Violet(UV) spectra as well as High Performance Liquid Chromatography (HPLC)retention times versus those of standards provided by the U.S. NationalCancer Institute (NCI), National Institutes of Health (NIH), Bethesda,Md. The identity of the Bryostatin-1 product was also confirmedindependently by Mass Spectral (MS) data as well as by ElementalAnalysis, Proton and Carbon Nuclear Magnetic Resonance (NMR), Infra Red(IR) spectroscopy, Differential Scanning calorimetry (DSC) and MeltingPoint.

Purification of Bryostatin-1 to 99.64% CP

Purification Procedure and Results:

An ethyl acetate extract of B. neritina (Sample C-021519#7), provided bythe National Cancer Institute (NCI), was used as the starting rawmaterials. A total of ˜57 g of the EA extract was dissolved indichloromethane (DCM) and assayed to determine presence of Bryostatin-1and other Bryoids. Turning now to FIG. 2, a HPLC Chromatogram of B.neritina EA crude extract is depicted. Labeled arrows indicate internaldesignations for Bryostatin-like compounds, which include B10, B12, andBryostatin 3 (B16), and Bryostatin-2 (Bryn-2) and Bryostatin-3 (Bryo-3).Bryostatin-1 (Bryo-1) elutes at 24.2 min. The designation B10 is thebryoid corresponding to the first bryoid of the present invention. Thedesignation of B14 will lead to the third and fourth Bryoids of thepresent invention.

Bryostatin-1 was purified from B. neritina EA crude extracts usingvarious chromatography resins as shown in FIG. 3. The initial steps(Step 1 and Step 2) were performed on Silica gel Active (100-200 μm),and the sample eluted with increasing concentrations of ethyl acetate inDCM. The silica purification steps are useful in removing some of thecolored components from the EA crude extract, the non-polar compounds(eluting at end of chromatographic run, FIG. 3), and eliminating themajority of the B16 peak.

Next, fractions containing Bryostatin-1 were purified on AmberchromCG71, which allowed for the elution of Bryostatin-like compounds withacidified methanol and water. This resin helps minimize the use ofchlorinated solvents that are harmful to the environment. CG71purification step removes the ‘X5’ peak eluting before Bryostatin-1. Italso served to minimize the impurities right before Bryostatin-1, mainlyB16.

Subsequent purification was performed using a combination of Amicon C1840 μm resin and two prep-C18 columns (2.5×2.5 cm, 10 μm column) Thisstep allowed for the further separation of B12 and Bryo-3 fromBryostatin-1, though there was still the presence of the ‘x5’ peak atthe shoulder of Bryostatin-1. Final crystallization step led to thepurification of Bryostatin-1 to >99% chromatography purity (CP), with a69% recovery from crude extract.

HPLC Monitoring:

During each purification step outlined in FIG. 3, Bryostatin-1 wasmonitored on a Luna C18(2) column (250×4.6 mm, 10 μm). Elution wasperformed at 80% acetonitrile acidified with phosphoric acid (ACNP) inan isocratic mode at a 2 mL/min flow rate. Column temperature was set at30° C.

Bryostatin-Like Compounds (Bryoids)

Bugula neritina was fractionated to produce Bryostatin fractions(Bryoids) that could serve as alternatives to Bryostatin-1. Thesefractions were purified and sent to LSU for in vitro analysis (Table 1).

TABLE 1 Amount (in mg) of each Bryoid in the Fractions as determined byHPLC¹ Fraction Sample B08 B10 B12 B14 Bryo-1 B16 % CP A: 101 B157 88.897.5 165 mL B: 102 B154 30.5 101.1 4.2 74.4 C: 103 B158 B12 60.4 100.86.7 49.3 350 mL D: 104 Bryo-2 100.9 94.4 E: 105 Bryo-AB 99.8 53.7 64.5F: 106 Bryo-3 98.3 12.1 72.1 G: 112 B16 APH 99.5 97.5 100311 CPcorresponds to Bryoid in bold

Efficacy of Bryostatin-1 Analogues (Bryoids) in Induction of s-APPαSecretion:

The efficacy of several Bryostatin-1 analogues (Bryoids) in induction ofs-APPα secretion is shown in FIG. 4. Except Fraction D, they all inducedsignificant release of s-APPα compared to Bryostatin-1. The bestalternative fraction to Bryostatin-1 is analogue E which corresponds tothe designation B16, which was identified as Bryostatin 3. Bioactivitywent in order from Fraction E (105)>G (112), F (106), C (103)>A (101), B(102)>D (104).

From the preliminary data, it appears that B12 or B14 can besignificantly more bioactive than Bryostatin-1. Since most fractionscontain two or more Bryoids, it is difficult to determine which one isresponsible for the bioactivity except for Fraction G, which containsB16 at >97.5% CP. The first bryoid composition of the present invention,B10, has significantly higher activity than Bryostatin-1, and it posesanother potential alternative Bryoid as a therapeutic.

HPLC Standardization:

Turning now to FIG. 5, which depicts a high performance liquidchromatograph of a bryoid mixture at 265 nm, various Bryoids are presentin the B. neritina EA crude extract.

The mixture of the Bryoids (B09, B10, B12, B14C, B14B, B16,Bryostatin-1, Bryostatin-2, and Bryostatin-3) were standardized for thepurpose of identification (based on retention time) and subsequentpurification of each Bryoid. A chromatogram depicting the results ofhigh performance liquid chromatography purification is depicted in FIG.6. These Bryoids contain similar UV patterns as Bryostatin-1 with amaximum wavelength at 265 nm as shown in FIG. 7 which depicts UV spectraof different bryoid at 265 nm.

Identification of each Bryoid was attempted on UV-HPLC and LC/MS/MSusing known standards and/or comparing to known masses in theliterature. This is important as previous preliminary in vitroexperiments (described above) have shown that these Bryoids may induces-APPα secretion at equal or even greater percentages than is observedfor Bryostatin-1.

Preliminary Characterization Based on LC/MS/MS

Characterization of the different Bryoids was performed using anLC/MS/MS API 2000 system equipped with a Shimadzu HPLC system. Q1 scanparameters were optimized for Bryostatin-1 m/z 427 [M+Na] (FIG. 8-1),scanning from 700 to 1000 amu. Mass spectrum scans of other fractionsare presented in FIGS. 8-2 through 8-7. A total of seven fractions wereanalyzed, which included individual Bryoids and mixture of Bryoids(Table 2).

TABLE 2 Bryostatin Analogue for Each Fraction Based on Mass MatchBryostatin Bryoid Mass + Na Mass [M] Match Based on Mass Fraction 101:Bryo-1 927.3 904.3 Bryostatin-1 Fraction 102: B12 and 911.4 888.4Bryostatin-3 B14 (Bryo-3) 925.4 902.4 None Fraction 103: B10 and 911.4888.4 Bryostatin-3 B12 897.2 874.2 None Fraction 104: Bryo-2 885.4 862.4Bryostatin-2 Fraction 105: B12 and 911.4 888.4 Bryostatin-3 Bryo-3Fraction 106: Bryo-3 911.4 888.4 Bryostatin-3 Fraction 112: B16 909.4886.4 None (tentatively identified as Bryostatin-3)

Discussion:

The LC/MS/MS data observed for Bryostatin-1 shows a peak at 927 Amu,which corresponds to the [M+NA], and what has been reported in theliterature (Manning et al., 2005). Mass spectral data on Bryostatin-1 to18 are summarized in Table 3. Based on the LC/MS/MS analysis performed,Fractions 104 and Fraction 106 were confirmed as Bryostatin-2 (863 Amu)and Bryostatin-3 (889 Amu), receptively.

Fraction 112 showed that B16 mass does not match any Bryoids reported inthe literature. Fractions 102, 103, and 105 showed a mass peak identicalto what was observed for Bryostatin-3. Both Fraction 102 and 105 containBryo-3 in their mixture, which would explain the 911 peak observed inthe LC/MS/MS. It is unclear why 911 Amu is seen in Fraction 103; thisindicates that B12 may have the same mass as Bryostatin-3 (889 Amu).This is supported by the fact that Fraction 105, containing both B12 andBryo-3, only showed a peak at 911 Amu. The 897 peak observed in Fraction103 could correspond to B10, though it does not match any of theBryostatin masses reported in the literature. The peak at 925 Amu inFraction 102 is also observed in Fraction 106.

TABLE 3 Mass Spectral Information on Bryostatin-1 to 18 (Manning et al.,2005) Monoisotopic M. M. + (Na⁺): M. M. ± (H₂): Group R1 monoisotopicGroup R2 monoisotopic Empirical Bryo. # mass 22.9892 2.0156 mass(attached) mass (attached) formula 1 904.4456 927.4348 902.4300 59.0133:139.0759: C₄₇H₆₈O₁₇ 906.4613 CH₃COO CH₃(CH₂)₂(CH)₄COO 2 862.4350885.4243 860.4194 17.0027: 139.0759: C₄₅H₆₆O₁₆ 864.4507 OHCH₃(CH₂)₂(CH)₄COO 3 888.4143 911.4035 886.3987 59.0133: 139.0759:C₄₆H₆₄O₁₇ 890.4300 CH₃COO CH₃(CH₂)₂(CH)₄COO 4 894.4613 917.4505 892.4456101.0602: 87.0446: C₄₆H₇₀O₁₇ 896.4769 (CH₃)₂CHCH₂COO CH₃(CH₂)₂COO 5866.4300 889.4192 864.4143 101.0602: 59.0133: C₄₄H₆₆O₁₇ 868.4456(CH₃)₂CHCH₂COO CH₃COO 6 852.4143 875.4035 850.3987 87.0446: 59.0133:C₄₃H₆₄O₁₇ 854.4300 CH₃(CH₂)₂COO CH₃COO 7 824.3830 847.3722 822.367459.0133: 59.0133: C₄₁H₆₀O₁₇ 826.3987 CH₃COO CH₃COO 8 880.4456 903.4348878.4300 87.0446: 87.0446: C₄₅H₆₈O₁₇ 882.4613 CH₃(CH₂)₂COO CH₃(CH₂)₂COO9 852.4143 875.4035 850.3987 87.0446: 59.0133: C₄₃H₆₄O₁₇ 854.4300CH₃(CH₂)₂COO CH₃COO 10 808.4245 831.4137 806.4088 101.0602: 1.0078:C₄₂H₆₄O₁₅ 810.4401 (CH₃)₃CCOO H 11 766.3775 789.3667 764.3619 59.0133:1.0078: C₃₉H₅₈O₁₅ 768.3932 CH₃COO H 12 932.4769 955.4661 930.461387.0446: 139.0759: C₄₉H₇₂O₁₇ 934.4926 CH₃(CH₂)₂COO CH₃(CH₂)₂(CH)₄COO 13794.4088 817.3980 792.3932 87.0446: 1.0078: C₄₁H₆₂O₁₅ 796.4245CH₃(CH₂)₂COO H 14 824.4194 847.4086 822.4037 101.0602: 17.0027:C₄₂H₆₄O₁₆ 826.4350 (CH₃)₃CCOO OH 15 920.4405 943.4297 918.4249 59.0133:155.0708: C₄₇H₆₈O₁₈ 922.4562 CH₃COO CH₃CH₂CHOH(CH)₄—COO 16 790.4139813.4031 788.3983 101.0602: 1.0078: C₄₂H₆₂O₁₄ 792.4296 (CH₃)₃CCOO H 17790.4139 813.4031 788.3983 101.0602: 1.0078: C₄₂H₆₂O₁₄ 792.4296(CH₃)₃CCOO H 18 808.4245 831.4137 806.4088 101.0602: 1.0078: C₄₂H₆₄O₁₅810.4401 (CH₃)₃CCOO H

Isolation of Bryostatin Analogues: B16 (98.5% CP) and B14B (93.4% CP)

Bryoid-like compounds, B16 and B14B, were purified from side-cutscollected from previous Bryostatin-1 purifications, and had been storedat 4° C. The bryoids' UV-spectra are identical to that of Bryostatin-1(FIG. 9).

HPLC Monitoring:

During purification, B16 and B14B were monitored on a Luna C18(2) column(250×4.6 mm, 10 μm). Elution was performed at 80% ACNP (acetonitrileacidified with phosphoric acid) isocratic mode, at a 2 mL/min flow rate.Column temperature was set at 30° C.

Purification Procedure and Results:

Fractions containing B16 and B14B were purified using two prep-C18columns (2.5×2.5 cm, 10 μm) and a semi-prep PFP column. Purification wasperformed with step-gradient using increasing concentrations of ACNP.Elution was monitored until each Bryoid was located mainly on individualcolumns. Columns were stripped using a fast gradient with ACNP, andfractions were assayed to determine concentration of each peak.

Bryoids B16 and B14 B can be separated successfully using the describedcolumn system. The use of both C18 and PFP column is necessary for theseparation of B16 from B14B, and partial purification of B14B from B14C.Peak labeled B14C is another bryoid that co-elutes with B14B, and can bebetter monitored when analyzed at 70% ACNP. Crystallization of both B16and B14B/C was possible by addition of MeOH to the Bryoid-containingfractions. A total of 212 mg of B16 crystals with 98.5% CP werecollected. A total of 108 mg of B14B/C at 93.4% CP was recovered andstored for future purification. B14B/C was subsequently separated intoB14B and B14C. The purified Bryoids were re-analyzed by LC/MS/MS. Theresults are summarized in Table 4.

TABLE 4 LC/MS/MS Analysis of Purified Bryostatin Analogues for EachFraction Based on Mass Match Bryoid Mass + Na Mass [M] Bryostatin MatchBased on Mass Bryostatin-1 927.3 904.3 Bryostatin-1 Bryostatin-2 885.4862.4 Bryostatin-2 Bryostatin-3 911.4 888.4 Bryostatin-3 B16 909.4 886.4None B10 897.4 874.4 None B12 911.5 888.9 Bryostatin-3 Isomer B14B 869.5846.6 None B14C 895.5 872.6 None

Biological Activities of Purified Bryoids:

Purified Bryoids at 10-⁹M are shown to increase alpha-secretase activityin SHSY-5Y neuroblastoma cells in FIG. 10. B3, B14B and B16 are shown toimprove the production of alpha-secretase over Bryostatin-1.

B10 is shown to improve the production of PKC-epsilon over Bryostatin-1in FIG. 11. B10 is shown to improve the production of PKC-delta overBryostatin-1 in FIG. 12. B10 is shown to improve the production ofPKC-alpha over Bryostatin-1 in FIG. 13.

NMR and Structural Characterization:

The three variants were compared by to bryostatin 1 and bryostatin 3 bytheir NMR ¹H and ¹³C resonances and connectivities (HSQC and HMBCspectra). All three variants distinctly had the ring closure at C22 ofbryostatin 3, and similar R1 and R3 sidechains (the OAc and the 8-carbon2,4-ene). The variations, relative to bryostatin 3, were:

B10: NMR showed loss of one methyl group from C18, matching the massdifference: Predicted C₄₅H₆₂O₁₇=874.4 (monoisotopic); obs B10 874.4. Theputative structure of B10 is depicted in FIG. 14.

B12 appears to be a stereoisomer: a number of protons in the vicinity ofthe 19-24 ring have modest changes in chemical shift; but theconnectivities show the same covalent structure as bryostatin 3, and ithas the same mass as bryostatin 3 (C₄₆H₆₄O₁₇=888.4). The most likelysite would be at C22, if the mechanism of ring closure was not perfectlystereoselective. Inversion at adjacent sites (19, 20, or 23) could alsoexplain the NMR changes, although these variations are not seen amongthe other bryostatins. The putative structure for B12 is depicted inFIG. 15.

In B16, the 26-OH has become a ketone. This change accounts for the 2 Daobserved mass difference between B16 (C₄₆H₆₂O₁₇=886.4) and Bryo-3(888.4). A bryostatin-3 26-ketone is known (Schaufelberger 1991).

These structures are suggested by the NMR data which is set forth in NMRspectra in FIGS. 16-18. FIG. 16 depicts the NMR spectra of Bryostatin-3.FIG. 17 depicts the NMR spectra of B10. FIG. 18 depicts the NMR spectraof B12 overlaid on the NMR spectra of Bryostatin-3.

Thus, we have disclosed embodiments of the present invention based onour present understanding of the best mode to make and use thesecompounds. Those skilled in the art will readily understand that suchpreferred embodiments are subject to alteration and modification andtherefore the present invention should not be limited to the precisedetails, but should encompass the subject matter of the claims thatfollow and their equivalents.

The invention claimed is:
 1. A method of making a first Bryoidcomposition comprising the steps of isolating a first Bryoid compositionfrom a source of Bryoids and purifying the first Bryoid composition in arange from 50% to a crystal forming purity wherein said first Bryoidcomposition has a molecular weight of approximately 896-898 Amu(Mass+Sodium) and 873-875 Amu (monoisotopic mass).
 2. The method ofmaking the first Bryoid composition of claim 1 wherein said first Bryoidhas a measured mass plus sodium of 897.2 Amu and a measured monoisotopicmass of 874.2 Amu.
 3. The method of making the first Bryoid compositionof claim 1 comprising the steps of: (i) extracting the first Bryoid withorganic solvents or with SuperFluids (near-critical and supercriticalfluids with or without cosolvents) solvents; (ii) partially purifyingthe first Bryoid by silica chromatography with organic solvents orSuperFluids chromatography; (iii) performing segmentation chromatographyon resulting polymeric resin to improve purity of the first Bryoid; (iv)performing C18 chromatography to further improve the purity of the firstBryoid; and (v) crystalizing the first Bryoid to still further improvethe purity of the first Bryoid.